Oxidative DNA Damage and Atherosclerosis: Reactive oxygen species (ROS) can damage cellular macromolecules such as DNA and proteins. DNA damage results directly from ROS, or from ROS-derived lipid-hydroperoxides that break down to form the alpha,beta-unsaturated aldehyde genotoxins, 4-oxo-2-nonenal, 4 hydroxy-2-nonenal, and 4,5-epoxy-2(E)-decenal. Lipid hydroperoxides are also formed enzymatically from 15-lipoxygenase (15-LOX) and from cyclooxygenase-1 (COX-1) and COX-2. We have recently shown that 4 oxo-2-nonenal and 4-hydroxy-2- nonenal are formed by two distinct pathways during the homolytic breakdown of the linoleic acid-derived prototypic omega-6 lipid hydroperoxide, 13(S)-hydroperoxyoctadecadienoic acid (HPODE). One pathway involves the intermediate formation of hydroperoxide-derived alkoxy radicals and also results in the formation of 4,5-epoxy-2(E)-decenal. The other pathway involves the intermediate formation of the potential genotoxin 4-hydroperoxy-2- nonenal, which then either undergoes a l-electron oxidation to give 4-oxo-2- nonenal or a 1-electron reduction to give 4-hydroxy-2-nonenal. 4,5-Epoxy-2( E)-decenal forms unsubstituted etheno-2?-deoxyadenosine adducts with DNA and so provides an important link with lipid peroxidation and DNA damage known to occur in human tissues. In recent studies, we made the surprising observation that vitamin C can stimulate the breakdown of lipid hydroperoxides to alpha,beta-unsaturated aldehyde genotoxins. Furthermore, the remarkable efficiency of this process suggests that vitamin C could give rise to significant levels of DNA-damage in vivo. If lesions derived from direct oxidative damage or lipid-hydroperoxide-mediated DNA damage are not repaired, replication can lead to mutations or apoptosis. Apoptotic cell death is involved in the pathogenesis of a variety of cardiovascular diseases, including heart failure, myocardial infarction and atherosclerosis. The ability to directly identify and quantify lipid hydroperoxide-mediated DNA damage will make it possible to assess the role of such lesions in atherosclerosis. We plan to determine the potency of each of the lipid hydroperoxide-derived bifunctional electrophiles in causing DNA-damage. Using in vitro models, the relative importance of lipid hydroperoxide mediated damage when compared with damage that occurs from ROS will be determined. The spectrum of adducts formed in 13(S)-HPODE and 9(R)-HPODE-treated cultured human endothelial cells pre-loaded with vitamin C will be characterized. DNA-adduct formation arising from endogenously generated 13(S)-HPODE and 9(R)-HPODE will also be assessed. The catalytic efficiency of COX-2-mediated linoleic acid oxidation to 13(S)-HPODE and 9(R)-HPODE is much greater than COX-1. However, normal endothelial cells only express COX-1. Therefore, COX-2 will be induced and the cells will then be treated with linoleic acid. The resulting HPODEs will be subjected to vitamin C-mediated decomposition. Cellular DNA will be isolated and the major adducts will be quantified. With these studies in hand, DNA-adduct formation in endothelial cells, leukocytes and urine from a mouse model of atherosclerosis will be examined. Finally, lipid hydroperoxide-derived DNA-adducts will be quantified in leukocytes and urine from normal subjects and from patients diagnosed with atherosclerosis. The relative importance of lipid hydroperoxide-derived and ROS-derived covalent modifications will then be assessed.